Without limiting the scope of the invention, its background is described in connection with fluorescence detection.
Without limiting the scope of the invention, its background is described in connection with fluorescence spectroscopy and fluorescence microscopy. Fluorescence is a phenomenon that has provided many useful applications and fluorescence spectroscopy is a rapidly developing and crucial component in the areas of flow cytometry, medical diagnostics, DNA sequencing, genetics and cellular and molecular imaging, and tissue and organ imaging.
During the last couple of decades, fluorescence based imaging has made incredible progress, becoming one of the most versatile and widely utilized visualization techniques in research and biomedical diagnostics. The quickly increasing availability of new dyes and fluorescent proteins as well as technological progress opens new ways for noninvasive studies of fundamental processes from gene expression, protein function, and protein-protein interactions to cellular and tissue processes [1,2]. In addition to outstanding successes in microscopy, fluorescence based imaging is gaining momentum as an imaging method for whole-body, in-vivo investigation of molecular processes in small animals [3-5]. The development of bright probes and highly sensitive detectors puts fluorescence among most sensitive detections frequently allowing study of nanomolar and picomolar concentrations [6].
A fundamental limitation for cellular and tissue imaging is sample autofluorescence, which is the fluorescence of, e.g., endogenous components of cells, tissue, and fixatives. The large variety of autofluorescence sources produces a broad emission that overlaps with the emission of typical fluorescent dyes used for labeling [7-9]. Typically the brightness of such natural components is relatively low, but the overwhelming abundance of them results in a significant contribution to the observed fluorescence signal, especially in tissue samples. It is very difficult to reduce the signal from autofluorescence without altering the probe and/or the biological system [10-12], thus one typically increases the concentration of the dye to a level at which the fluorescence of the dye dominates the overall signal. Such a large increase in dye concentration is not always possible for various physiological reasons, and in many cases it may interfere with the biological process one wants to investigate. In cases where background is a problem, an increase in excitation intensity is not an option, since it always leads to a proportional increase in both background intensity and signal intensity. Despite recent advances, a need remains for overcoming sample autofluorescence.